Spatial genomics reveals cholesterol metabolism as a key factor in colorectal cancer immunotherapy resistance.

Immune checkpoint inhibitors (ICIs) have transformed the treatment landscape across multiple cancer types achieving durable responses for a significant number of patients. Despite their success, many patients still fail to respond to ICIs or develop resistance soon after treatment. We sought to identify early treatment features associated with ICI outcome. We leveraged the MC38 syngeneic tumor model because it has variable response to ICI therapy driven by tumor intrinsic heterogeneity. ICI response was assessed based on the level of immune cell infiltration into the tumor - a well-established clinical hallmark of ICI response. We generated a spatial atlas of 48,636 transcriptome-wide spots across 16 tumors using spatial transcriptomics; given the tumors were difficult to profile, we developed an enhanced transcriptome capture protocol yielding high quality spatial data. In total, we identified 8 tumor cell subsets (, proliferative, inflamed, and vascularized) and 4 stroma subsets (, immune and fibroblast). Each tumor had orthogonal histology and bulk-RNA sequencing data, which served to validate and benchmark observations from the spatial data. Our spatial atlas revealed that increased tumor cell cholesterol regulation, synthesis, and transport were associated with a lack of ICI response. Conversely, inflammation and T cell infiltration were associated with response. We further leveraged spatially aware gene expression analysis, to demonstrate that high cholesterol synthesis by tumor cells was associated with cytotoxic CD8 T cell exclusion. Finally, we demonstrate that bulk RNA-sequencing was able to detect immune correlates of response but lacked the sensitivity to detect cholesterol synthesis as a feature of resistance.